Medicago-Sinorhizobium GeneChip procedures
After the supplementary protocol of Barnett et al. 2004. PNAS 101: 1663616641 and the Affymetrix expression_analysis_technical_manual.pdf,
but using different RNA isolation and DNA removal protocols.
Reagents and items needed:
Forceps for removing nodules
Liquid nitrogen (LN2)
Dry ice
Mortar & pestle
TRI REAGENT (Molecular Research Center, Inc #TR 118)
Sturdy polypropylene microcentrifuge tubes
RNase-away (Invitrogen, #10328011)
RNase-free water
RNase-free water, pH adjusted to 7.0 with NaOH
100 bp DNA ladder (New England Biolabs #N3231S)
Turbo DNA-free kit (Ambion #AM1907)
DNAse I (Pierce #27-0514-0 or Fisher #PI-89835)
One-phor-all 10X buffer (USB #78331)
Qiaquick PCR Purification Kit + buffer PB for cDNA cleanup (Qiagen #28104)
Random primers (Hexamers, 75 ng/µl, Invitrogen #48190011)
Superase Inhibitor (Ambion #AM2694)
Superscript III (200 unit/µl Invitrogen #18080044)
with 100mM DTT buffer and 5x first-strand buffer: 250 mM Tris pH 8.3, 375 mM
KCl, 15 mM MgCl2
10 mM dNTPs (Invitrogen #N80800078)
I N NaOH
I N HCl
GeneChip DNA Labeling Reagent (Affymetrix #900542)
Terminal Deoxynucleotidyl Transferase, Recombinant (Promega #M1871)
Terminal Transferase, Recombinant, is supplied in 50mM potassium phosphate (pH
6.4), 100mM NaCl, 1mM β-mercaptoethanol, 0.1% Tween® 20 and 50% glycerol
Terminal Transferase 5X Buffer: 500mM cacodylate buffer (pH 6.8), 5mM CoCl2
and 0.5mM DTT
Poly-A RNA controls (Affymetrix #900433)
All supplies and reagents must be RNAse-free at all steps until cDNA is made.
Gels for DNA may be 1.5% agarose in 0.5x TBE buffer. The tracking dyes will have
apparent molecular weights of ~4kb (xylene cyanol FF), ~ 300bp (bromophenol
blue), ~50bp (Orange G). Choose dyes that do not obscure your sample. RNA may be
run on the same gels, although bands will not be a sharp as for RNA run on
formaldehyde gels.
1
Isolation of Nodule RNA
Grow plants as desired. At least 800 mg of nodules are needed for a single
Affymetrix GeneChip. Before harvest, label 50-ml centrifuge tubes and pack in dry
ice. To excise nodule material, pinch the nodule with forceps at the nodule-root
junction. Periodically add more liquid nitrogen to the tube while harvesting. Keep
tubes on dry ice until nodule harvest is completed, then cap and transfer to liquid
nitrogen for transport. Tissue may be stored at -80°C or used immediately. See
Table 1 below for the plant samples used in this experiment.
A. Total RNA Extraction:
1. Freeze mortar and pestle completely before adding nodules
(pack mortar in dry ice; after mortar and pestle are cold add LN2)
2. Add nodule tissue from both samples to be pooled (2 plants worth of nodules),
and grind to fine powder (repeatedly adding LN2 to keep frozen)
3. Add 3mL TRI Reagent directly to frozen mortar/pestle and tissue, and grind to a
fine powder; remove mortar from dry ice bed and continue grinding as material
thaws. This is period of maximum RNA extraction.
4. Split homogenized tissue into 3 tubes, each with ~1.0 mL TRI Reagent mix and
proceed per tube:
5. Centrifuge TRI Reagent preps at 12,000 g for 10 minutes at 2-8 C to precipitate
tissues. RNA is in supernatant. Transfer cleared homogenate to a fresh tube.
6. Incubate tubes at room temp for 5 minutes.
7. Phase separation: add 0.2 mL chloroform to each tube (0.2 mL per 1 mL TRI
Reagent) & shake vigorously for 15 seconds. Incubate for 2-3 minutes at room temp.
8. Centrifuge at 12,000 g for 15 minutes at 2-8° C for phase separation (RNA is in
upper, aqueous phase). Transfer aqueous phase to fresh tube (~ 900 L aqueous
recovered).
9. RNA precipitation: Add 0.50 mL isopropanol to aqueous phase (0.5 mL per 1 mL
TRI Reagent).
10. Incubate samples at 15-30° C for 10 minutes, then centrifuge at 12,000 g for 10
minutes at 2-8° C. RNA precipitate forms gel-like pellet on bottom/sides of tube.
11. Remove supernatant and wash pellet with cold 1 mL 75% ethanol. Vortex and
centrifuge at 7,500 g for 5 minutes at 2-8° C. Decant ethanol and invert tube on clean
kimwipe (removing as much EtOH as possible – carefully tap tubes on kimwipe
repeatedly).
12. Briefly dry the RNA pellet by air-drying at room temp for 3-5 minutes in the
laminar flow hood. (don’t overdry, as this will reduce RNA solubility). Sniff to make
sure EtOH is gone.
13.Resuspend in 50 μl RNase-free water, incubate 10 min. at 55-60° C and store RNA
at -80° C to minimize degradation.
2
Quantify RNA spectrophotometrically. Ideally, 260/280 ratio should be >1.8.
Run RNA gel. Hope to see 4 clear bands for rRNAs, little smearing and little
degradation (low molecular weight smear). See Figure 1 for example agarose gel.
B. Turbo DNA-free kit for genomic DNA contamination of RNA samples.
(product Ambion AM1907, protocol modified from manufacturer’s)
Transfer RNAs to 0.5 mL tubes, to facilitate removal of supernatant.
For 50 µl sample of RNA:
1. Add 5 µl 10X Turbo DNase Buffer to the RNA and mix gently.
2. Add 1.5 µl DNase and mix gently. Incubate for at 37 ° C for 30 minutes (in shaker).
3. Add 1.5 µl more enzyme, and repeat incubation.
3. (Always resuspend reagent by flicking/vortexing tube before dispensing). Add 10
µl DNase inactivating reagent and mix well.
4. Incubate 5 min. at room temperature, mixing occasionally (2-3 times) to redisperse the DNase Inactivation Reagent. (Make sure room is between 22-26° C or
can decrease activity of this step, leaving DNase in the reaction).
5. Centrifuge at 10,000 x g for 1.5 minute and carefully transfer the RNA
(supernatant) to a fresh tube (be sure to avoid the pelleted inactivation reagent.
3
C. cDNA synthesis.
As per Affymetrix expression_analysis _technical_manual.pdf, except using
Superscript III and inactivating this enzyme for 15 minutes at 70 C.
Dilute poly-A RNA controls (Affy #900433)
1. Make 2 serial 1:20 dilutions of stock RNAs (for use with the 100 (midi) Format
Medicago chip)
2. Add 2 μl in each 30 l reaction below
Allow a minimum of 30 µg of RNA per chip (Barnett et al. 2004). Perform
reactions in triplicate, using 10 µg of RNA in each. cDNA yields may be less
3. Prepare the following mixture in new PCR strip-tubes:
Primer Hybridization Mix
Components Volume/amount Final concentration
Total RNA
10 µg
0.33 µg
Random
10 µl
25 ng/µl
primers
Hexamers
(75 ng/µl)
(Invitrogen)
Diluted polyA RNA
controls
(Affymetrix
#900433)
Nucleasefree H2O
2 µl
Total volume
30 µl
variable
Up to 30 µl
4. Using a thermal cycler, incubate the RNA-Primer mix at 70°C for 10 min followed
by 25°C for 10 min and then chill to 4°C.
5. Prepare triplicates of the reaction mix for cDNA synthesis and add to the annealed
RNA-primer mix.
4
cDNA Synthesis Reaction Mix
Components
Volume/amount
5X first strand buffer
12 µl
(Invitrogen)
100 mM DTT
6 µl
(Invitrogen)
10 mM dNTPs
3 µl
(Invitrogen)
SUPERase Inhibitor
1.5 µl
(20 unit/µl)
(Ambion, Austin TX)
SuperScript III
7.5 µl
(200 unit/µl)
(Invitrogen)
Annealed RNA-primer mix 30 µl
Total volume
60 µl
Final concentration
1X
10 mM
0.5 mM
0.5 unit/µl
25 unit/µl
6. Incubate the reaction at 25°C for 10 min, 37°C for 60, then 42°C for 60 min.
7. Inactivate the enzyme at 70°C for 15 min and hold at 4°C.
Removal of RNA
8. Add 20 µl of 1 N NaOH and incubate at 65°C for 30 min.
9. Add 20 µl of 1 N HCl to neutralize.
5
D. Purification and Quantification of cDNA Synthesis Products
(Modified from Qiagen QIAquick PCR Purification Kit protocol. Qiaquick spin
columns are the same in Qiagen gel extraction or PCR purification kits)
All spins at 12000 rpm (~15000 g) in an Eppendorf microfuge:
1. Add 500 μl of Buffer PB to cDNA sample (100 μl) and mix.
Make sure to use binding buffer PB; avoid any buffer with pH-indicator dye, which
will interfere with microarray reactions.
2. Put sample on Qiaquick spin column (in collection tube) and spin for 60 seconds.
3. Discard flow-through and put column back in the same tube.
4. To wash, add 750 μl Buffer PE to column and spin 60 seconds.
5. Discard flow-through and place column back in the same tube. Spin again to
remove residual ethanol.
6. Place column in clean microcentrifuge tube.
7. Add 20 μl H20 of pH>~7.0. Let stand for 60 seconds, then spin for 60 seconds to
elute DNA.
8. Repeat step 7 with same collection/microfuge tube (~40 μl elution volume total).
9. Leave tubes, caps open, on bench top at room temp for 20 minutes to remove
residual ethanol.
Quantify the purified cDNA product by 260-nm absorbance (1.0 A260 unit = 33
µg/ml of single-strand DNA). For a microcuvette use 1 μl in 50 μl water.
Visualize cDNA on agarose gel (see Figure 2)
6
E. cDNA Fragmentation
cDNA concentrations were adjusted for use with our GeneChip. Terminal labeling
was doubled for root nodules and plant roots to ensure excess label for these
reactions (Barnett et al. 2004). Use total of 12 µg cDNA for root nodule chips.
1. Prepare the following reaction mix:
Fragmentation Reaction:
Components
Volume/amount
Concentration
10X One-Phor-All buffer
5 µl
1X
(USB part 78331, ask for
Affymetrix price)
cDNA
40 µl
12 µg
DNAse I (see note)
x µl
x unit/µg of cDNA
Nuclease-free H2O
Up to 50 µl
Total volume
50 µl
Note: The amount of DNase I depends on its titer which must be checked (see below).
Dilute DNase I to x unit/µl in 1X One Phor-All-Buffer. Use immediately and do not
store.
2. Incubate the reaction at 37°C for 10 min. Inactivate the enzyme at 98°C for 10
min.
3. Add fragmented cDNA directly to the terminal labeling reaction. Alternatively, the
cDNA can be stored at -20°C and labeled later.
4. To examine fragmented cDNA, load 200 ng on a 1.5% agarose gel using an Orange
G load buffer and stain with sybr GOLD (details are given in Affymetrix manual).
Ethidium bromide may be used but will give very faint fluorescence with singlestranded DNA (see Table 2 and Figure 3 below).
7
The remainder of the protocol was performed by Thanh Nguyen at the
University of Toronto CAGEF processing facility, following the Affymetrix
protocol for 100 Format (Midi) GeneChips with double terminal labeling and a
hybridization temperature chosen to accommodate plant and bacterial
genomes (Barnett et al 2004). Details of the hybridization and wash protocols
are specific to the DNAs and the CAGEF Facility. See Affymetrix manual for
suggestions.
Terminal Labeling
Reagents:
GeneChip DNA Labeling Reagent, Affymetrix p/n 900542 – to label the 3’termini of the
fragmentation products.
Terminal Deoxynucleotidyl Transferase, Recombinant, Promega# M1871 including
- Terminal Deoxynucleotidyl Transferase, Recombinant, and
- Terminal Transferase 5X Buffer
1.
Prepare the following Reaction Mix:
edited from Section 3. Prokaryotic Sample and Array Processing / Terminal Labeling
Table 3.1.5 = terminal Label Reaction (page 3.1.12)
Terminal Label Reaction 2X (Nodule and Plant)
Components
x1 (μl)
cDNA = 49μl
sample 1-8
x9
5x Reaction Buffer
GeneChip DNA Labeling Reagent, 7.5mM
Terminal Deoxynucleotidyl Transferase
Fragmentation cDNA product (nodule = 12ug)
Water
16.00
4.00
4.00
47.75
8.25
16.00
4.00
4.00
49.00
7.00
144
36
36
Total Volume = 80μl
80.00
80.00
279
take 31μl
63
2. Incubate the reaction @ 37oC for 60minutes.
3. Stop the reaction by adding 2μl of 0.5M EDTA.
Alternatively, the reaction mixture may be stored @-20oC.
8
Prokaryotic Target Hybridization
1.
Prepare the following solution mix:
Hybridization Cocktail see table in the GeneChip Expression 3’Amplification
The reason to scale down the volume of the hybridization cocktail to 210μl is to save on
the amount of cDNA input - due to the original lowcDNA yield.
2X Hybridization Mix
3nM B2 Control
20X Control (65oC)
DMSO
Fragmented + Labeled cRNA
Water
Total Volume = 210μl
1
105
3.5
10.5
21
70
210
x9
945
31.5
94.5
189
1260
take 140μl per sample
Note:


2.
3.
4.
5.
Heat 20x control at 65 C for 5 mins before adding to the cocktail
Protocol could be stopped here. Store samples @ -20oC.
Equilibrate probe array to RT immediately before use.
Add 200μl of the Hybridization Cocktail to the probe array.
Cover the septa with Tough Spots
Hybridize @ 48oC, 60rpm, for 16h. in an Affymetrix Hybridization Oven 640.
9
Probe Array Washing and Staining
-prime the Fluidics Station
-the next day, transfer hybridization samples from gene chipslabeled microtubes
store -20oC
-fill Chips with 250μl of WashA – to prevent drying
Using Hybridization +Wash+Stain Kit pn. 900720
-aliquot 600μl of Stain1 for tube 1
- aliquot 600μl of Stain2 for tube 2
-aliquot 800μl of Array Holding Buffer for tube 3.
Fluidics Scripts:
-use FS450_001 for Medicago Chip
In case of bubble in the chip after staining, 800μl of the Wash A could be used to refill
tube 3.
10
DNase I Titer
Each lot no. of DNase I can vary greatly. To titer DNase I, determine the amount of
DNase I needed to fragment 1 µg of cDNA to sizes between 50 and 200 bases.
Choose between 0.2 and 1.1 DNase I units/µg cDNA to start.
Prepare the reaction mix as above with less cDNA to start:
Components
Volume/amount
Concentration
10X One-Phor-All buffer
5 µl
1X
(USB part 78331, ask for
Affymetrix price)
cDNA
40 µl
3-7 µg
DNAse I (see note)
x µl
x unit/µg of cDNA
Nuclease-free H2O
Up to 50 µl
Total volume
50 µl
TRIAL RUN TITRATION REACTION:
For initial chip, titer DNAse using 5 reactions (range from 0.2 – 1.1 Units / µg):
Components
Volume/amount
Concentration
10X One-Phor-All buffer
0.72 µl
1X
(USB part 78331, ask for
Affymetrix price)
cDNA
5.78 µl
1 µg
DNAse I (see note)
x µl
x unit/µg of cDNA
Nuclease-free H2O
Up to 7.6 µl
Total volume
7.6 µl
DNAse titration reactions. Gel stained with ethidium bromide, BPB and Orange G
stains partially obscure fluorescence
100bp
cDNA
units / µg cDNA
0.2
0.6
0 .8
1.1
100bp
0.2
units / µg cDNA
0.4
0.6
0.8
1.1
11
Tables and Figures
Table 1. RNA extractions of nodule tissue. 2 plants from each treatment
combination were pooled in a single RNA extraction.
Plant
Treatment Block Plant
Rhizobium Date
Sample
genotype genotype
extracted
3/124
A
1
Naut1
SalsB
37/75
B
1
Naut1
SalsC
cDNA
on
chip
5/25/2009 bot1446 10.2
ug
5/25/2009 bot1447 10.5
81/116 C
18/62 D
1
1
Sals4
Sals4
SalsB
SalsC
5/25/2009 bot1448 11.1
5/25/2009 bot1449 8.5
Plant
Treatment Block Plant
Rhizobium Date
Sample
genotype genotype
extracted
Chip
name
10/67 A
79/125 B
2
2
Naut1
Naut1
SalsB
SalsC
cDNA
on
chip
6/15/2009 Bot1489 7.3
6/15/2009 Bot1490 6.6
26/47
19/68
2
2
Sals4
Sals4
SalsB
SalsC
6/15/2009 Bot1491 4
6/15/2009 Bot1492 6.1
C
D
Plant
Treatment Block Plant
Rhizobium Date
Sample
genotype genotype
extracted
Chip
name
Chip
Name
25/80
11/58
A
B
3
3
Naut1
Naut1
SalsB
SalsC
cDNA
on
chip
9/11/2009 Bot1493 8.2
9/11/2009 Bot1496 6.0
4/52
5/69
C
D
3
3
Sals4
Sals4
SalsB
SalsC
9/11/2009 Bot1495 9.4
9/11/2009 Bot1494 10.2
12
Table 2: Example of fragmentation reaction for sample 146.
Components
10X One-Phor-All buffer
(USB part 78331, ask for
Affymetrix price)
cDNA
DNAse I (see note)
Nuclease-free H2O
Total volume
Volume/amount
5 µl
Concentration
1X
44 µl
(0.75 µl (10 unit / µl))
Up to 50 µl
50 µl
12.5 µg
(0.6 unit/µg of cDNA)
13
Figure 1: Example gel of total RNAs. RNAs were loaded on a 1.5 agarose 0.5x TBE gel
and stained with ethidium bromide. Bands for the 4 rRNAs are visible for all samples.
14
Figure 2: Visualization of cDNA on agarose EtBr gel. Ran 3 µl cDNA + 2 µl loading
buffer.
100 bp
146_1
146_2
162_1
15
Figure 3. Results of fragmentation reaction for sample 146. 2 µl each of cDNA and
fragmentation reaction in Orange G load buffer were run on a 1.5% agarose, 0.5x
TBE gel and stained with ethidium bromide. The fragmented DNA-ethidium
bromide complex was faint but clearly visible, and is just visible in the photograph.
1
2
3
100 bp cDNA frag
16